Studies on the metabolism of glutathione indicate several functions of glutathione, which include catalysis, metabolism, transport and cellular protection against reactive oxygen compounds, toxic compounds, and free radicals (see FIG. 1). Glutathione is synthesized intracellularly by the consecutive actions of .gamma.-glutamylcysteine and glutathione synthetases. Transport (export) of glutathione is characteristic of many cells and is of great importance in protection of cell membranes. Intracellular glutathione disulfide is a substrate of glutathione reductase (which uses reduced pyridine nucleotide) to provide cells with a reducing environment; thus under normal conditions less than 1% of total cellular glutathione is glutathione disulfide and little glutathione exists as mixed disulfides with proteins or low molecular weight thiols. Glutathione peroxidase catalyses the glutathione-dependent reduction of hydrogen peroxide and of other peroxides which are cytotoxic. Certain glutathione S-transferase catalyze the reduction of lipid peroxides; glutathione reduces the oxy radical form of .alpha.-tocopherol which is also involved in cell membrane protection. Free radicals are destroyed by glutathione in an apparently nonenzymatic reaction.
Glutathione is a specific cofactor for several enzymes such as glyoxalase, maleylacetoacetate isomerase, formaldehyde dehydrogenase, dehydrochlorinases, and prostaglandin endoperoxidase isomerases.
Glutathione reacts enzymatically (glutathione S-transferases) and nonenzymatically to form glutathione S-conjugates with endogenous compounds (e.g., leukotriene A, estrogens, prostaglandins) and exogenous compounds (e.g., bromobenzene, melphalan, etc.). These glutathione S-conjugates are metabolized via the mercapturic acid pathway. This pathway is a route of drug detoxication.
Depletion of cellular glutathione by inhibitors of .gamma.-glutamylcysteine synthetase should make tumor cells more susceptible to many anti-cancer drugs and to radiation. By depleting glutathione levels the destructive effects of reactive oxygen intermediates and of free radicals is used to advantage. Most normal cells have a large excess of glutathione, but tumor cells and parasites may have levels of glutathione that are close to that required for survival. Thus depletion of glutathione leads to selective sensitization of cells under hypoxic conditions (thereby decreasing the oxygen enhancement ratio). Certain tumor cells that have become resistant to drugs such as phenylalanine mustard, develop higher glutathione levels than the sensitive cells. Treatment of the resistant cells with the .gamma.-glutamylcysteine synthetase inhibitor, buthionine sulfoximine, depletes glutathione levels and leads to a reversal of drug resistance. In studies on human ovarian tumors, it was found that resistance to one drug led to a cross resistance to other anti-cancer drugs and radiation; thus increased glutathione levels seems to be a factor in cellular resistance.
It was recognized that depletion of cellular glutathione by treatment with the sulfoximine inhibitors of .gamma.-glutamylcysteine synthetase might make tumor cells more susceptible to the effects of radiation and of certain chemotherapeutic agents Meister et al, Cancer Treat. Rep., 1979, 63:1115-1121. In this approach, the destructive effects of reactive oxygen intermediates, such as hydrogen peroxide and free radicals are used advantageously. Glutathione functions to protect cells against toxic compounds of both endogenous and exogenous origin. It destroys reactive oxygen compounds and free radicals, and forms conjugates with certain compounds that have electronegative moieties. That cells depleted of glutathione become more sensitive to radiation and to the effects of certain toxic compounds is in accord with these conclusions.
In studies on three human lymphoid cell lines, the cells were depleted of glutathione to about 5% of the control levels by incubation in media containing buthionine sulfoximine. The glutathione levels of the cells decrease progressively over a period of 50 hours. Although cells with a level of 0.09 mM glutathione (4% of controls) were 85% viable, further decrease in glutathione level was associated with marked loss of viability. Cells that had 4-5% of the control levels of glutathione were much more sensitive than were control cells to the effects of .gamma.-radiation. About 50% of the intracellular glutathione that disappeared from cells of the CEM line was found in the medium. Although the medium was supplemented with L-serine plus borate to inhibit cellular .gamma.-glutamyl transpeptidase activity, such inhibition was incomplete so that less than theoretical recovery of the exported glutathione was found. The rate at which cellular glutathione is depleted is determined by the rate of glutathione export. Cells that export glutathione very slowly exhibit a very slow decline in cellular glutathione level when treated with buthionine sulfoximine. Depletion of cellular glutathione of human lymphoid cells to about 5% of the control levels led to a marked increase in sensitivity to radiation. Similar results have been obtained on cultured human lung carcinoma and on other tumor cells Biaglow et al, Radiat. Res., 1983, 95:437; Mitchell, et al, Radiat. Res., 1983, 96:422. Suspension of V79 cells in media containing buthionine sulfoximine led to selective e sensitization to radiation under hypoxic conditions and to a decrease of the oxygen enhancement ratio Biaglow et al, Radiat. Res., 1983, 95:437; Guichard et al, Proc. Radiat. Res. Soc., 1983, Abstr. Dc10. A considerable literature has developed on the sensitization of various types of cells, especially tumor cells, to radiation and to drugs by use of buthionine sulfoximine to deplete cellular glutathione (see, for example Midander et al, Radiosensitization Newsletter, 1984, 3(1):1-2; Biaglow et al, Radiat. Res, 1983, 95:437; Mitchell et al, Radiat. Res., 1983, 96:422; Guichard et al, Proc. Radiat. Res. Soc., 1983, Abstr. Dc10; Russo et al, Int. J. Radiat. Oncol. Biol Phys., 1986, 12-1347-1354; Shrieve et al, Radiat. Res., 1985, 102:283-294; Yu et al, Int. J. Radiat. Onol. Biol. Phys., 1984, 10:1265-1269; Clark et al, Int. J. Radiat. Oncol. Biol. Phys., 1986, 12:1121-1126; Arrick et al, J. Biol. Chem., 1982, 257:1231; Russo et al, Cancer Treat. Rep., 1985, 69:1293-1296. Most of thee studies were carried out on cultured cells or cell suspensions. Studies on mice bearing B.sub.16 melanomas have also shown that treatment of the animals with buthionine sulfoximine sensitizes the tumors to radiation. In these studies, the tumors were implanted in the footpads of mice and allowed to grow to a size of 250 mm.sup.3. The mice were given buthionine sulfoximine and were irradiated when the glutathione content of the tumors was about 20% of that of untreated controls. A significant decrease in tumor size and a significant increase in longevity was found.
Studies on mice depleted of glutathione by administration of buthionine sulfoximine led to increased sensitivity (a decrease of about 80% in the LD.sub.50 value) to acetaminophen. Studies on the oxidative cytolysis of several tumor cell lines by glucose oxidase, and by activated macrophages and granulocytes in the presence of phorbol myristate acetate, showed that depletion of glutathione by incubation in medium containing buthionine sulfoximine enhanced the degree of cytolysis Arrick et al J. Biol. Chem., 1982, 257:1231. The recovery of tumor cell resistance to peroxide was closely correlated with the resynthesis of cellular glutathione. Glutathione depletion sensitizes certain tumors to the effects of sulfhydryl reactive drugs Arrick et al, J. Clin. Invest., 1983, 71:258.
In a study in which six mice infected with Trypanosoma brucei were treated with buthionine sulfoximine, two mice were apparently cured and four mice survived significantly longer than did untreated infected controls Arrick et al, J. Exp. Med., 1981, 153:720. This organism apparently does not contain catalase and the findings therefore suggest that depletion of glutathione can be an effective approach for the destruction of cells that lack catalase; thus, the glutathione peroxidase system is the only available mechanism for destruction of hydrogen peroxide.
In early studies on the effects of certain antineoplastic agents, it was observed that tumors developed increased levels of thiols. Vistica and collaborators examined the toxicity of phenylalanine mustard toward resistant and sensitive mouse L1210 leukemia cells. The dose of phenylalanine mustard needed to kill the most resistant cells was substantially higher than that required to kill the most sensitive cell lines. It was shown that the resistance to phenylalanine mustard was not related to differences in uptake or efflux of the drug, but rather to the cellular level of glutathione. It was also observed that the resistant cells converted phenylalanine mustard to a non-toxic hydroxy derivative in a glutathione-dependent dehydrochlorination reaction. [Presumably this is similar to that found in houseflies that have developed resistance to the insecticide DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane)]. Treatment of the resistant cells with buthionine sulfoximine led to resensitization of the tumor cells to phenylalanine mustard. In studies on mice bearing resistant tumors, sensitization of the tumors to the effects of phenylalanine mustard was achieved by continuous intraperitoneal infusion of buthionine sulfoximine; and increase in the life span of these animals was observed. It was also found that phenylalanine mustard-resistant cells were able to convert more of the mustard to its nontoxic hydroxy derivative than were the mustard-sensitive cells.
Human ovarian cancer cells that are resistant to phenylalanine mustard also exhibit increased levels of glutathione Green et al, Cancer Res., 1984, 44:5427-5431; Behrens et al, Proc. Amer. Assoc. Can. Res., 1984, 35:336; Hamilton et al, Cancer Res., 44:5286-5290; Louie et al, Biochem. Pharmacol., 1985, in press; Hamilton et al, Biochem. Pharmacol., 1985, 34:2583-2586; Ozols et al, "Glutathione Depletion with Buthionine Sulfoximine: Potential Clinical Applications" in Biochemical Modulators: Experimental and Clinical Approaches; Valeriote et al, Eds.; Martinus Nijhaus: Boston, 1986, pp. 277-294.
Reversal of resistance can be achieved in such cells by depletion of glutathione by use of buthionine sulfoximine. It is of interest that resistance of these tumors to phenylalanine mustard was accompanied by resistance to other drugs such as adriamycin; in addition, the phenylalanine mustard-resistant cells are also resistant to radiation. Glutathione depletion significantly increases the sensitivity of the resistant cells to the drugs and to radiation. The common denominator that underlies such resistance is increased cellular levels of glutathione. It should be mentioned, however, that there is evidence that other factors play a role in resistance and that some types of resistance to drugs are not associated with elevation of glutathione levels. The effects observed on phenylalanine-mustard resistant human ovarian tumor cells grown in nude mice, i.e., reversal of resistance after treatment with buthionine sulfoximine, have led to consideration of the use of buthionine sulfoximine in the therapy of humans with ovarian tumors; Ozols et al, supra.
The collected data indicate that depletion of glutathione by administration of buthionine sulfoximine may be useful in cancer chemotherapy and in radiation therapy of cancer Arrick et al, Cancer Res., 1984, 44:4224-4232. Depletion is effective when the tumor cells and normal cells have significantly different requirements for glutathione. Many normal cells probably have an excess of glutathione whereas certain tumors and parasites contain levels of glutathione that are close to the minimal required for survival. The response of some cells to anticancer agents and other toxic compounds (see, for example Lipke et al, J. Biol. Chem., 1959, 254:2131-2128; Goodchild et al, Biochem. J., 1970, 117:1004-1009; Balabaskaran et al, Biochem. J., 1970, 117:989-996; Dinamarca et al, Arch. Biochem. Biophys. 1971, 147:374-383) leads to increased levels of glutathione (probably due to induction of the synthetases), which are responsible for one type of drug resistance. As one would expect, development of resistance associated with elevated glutathione levels to one drug would lead to resistance to others and to radiation.
Tumors with low levels of catalase and which therefore depend upon the activities of glutathione peroxidase and of glutathione S-transferases for destruction of peroxides, would be expected to become less viable during glutathione depletion. Depletion of glutathione in a tumor cell that lacks catalase altogether would be expected to lead to death of the cell, but normal cells, which have catalase, might not be significantly affected. Tumors relatively resistant to radiation and that have high levels of glutathione would be expected to become more sensitive to radiation after cellular levels of glutathione are decreased.
The following references inter alia describe buthionine sulfoximine and related compounds and their uses. These compounds and their described uses appear to be relevant prior art:
Griffith et al, J. Biological Chem., 1979, 254:1205-1210; PA0 Griffith et al, J. Biological Chem., 1979, 254:7558-7560; PA0 Meister et al, Cancer Treat. Reports., 1979, 63.:1115-1121; PA0 Griffith, J. Biological Chem., 1982, 257:13704-13712; PA0 Dethmers et al, Proc. Nat'l. Acad. Sci. USA, 1981, 78:7492-7496; PA0 Meister, Science, 1983, 220:472-477; PA0 Meister, Hepatology, 1984, 4:739-742; PA0 Meister, Nutritional Rev., 1984, 42:397-410; PA0 Meister, Current Topics in Cellular Regulation, 1985, 26:383-394; PA0 Griffith et al, Proc. Nat'l Acad. Sci. USA, 1985, 82:4668-4672.